This study aims to explore which radicals dominate sodium nitroprusside (SNP)-induced cytotoxicity in human hepatocellular carcinoma (HCC) cells (HepG2 and Hep3B). Exposure of SNP to cell medium produced abundant nitric oxide (NO), superoxide anion (O2·−), hydrogen peroxide (H2O2) and iron ions. SNP potently induced caspases activation, mitochondrial membrane permeabilization and apoptosis in HCC cells. In Hep3B cells, pretreatment with NO scavenger (PTIO) did not prevent SNP-induced cytotoxicity. However, in HepG2 cells, SNP-induced cytotoxicity was prevented significantly by pretreatment with PTIO and O2·− scavenger, and especially was almost completely blocked by pretreatment with FeTPPS (peroxynitrite scavenger). In contrast, although H2O2 scavenger potently scavenged SNP-induced H2O2 production, it did not prevent SNP-induced cytotoxicity in HepG2 cells. In addition, pretreatment with DFO (iron ions chelator) and iron-saturated DFO respectively completely prevented SNP-induced cytotoxicity in HepG2 cells. Collectively, peroxynitrite from the reaction between NO and O2·− elicited from SNP dominates the SNP-induced apoptosis of HepG2 cells, in which both iron ions and H2O2 are not involved.

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Exact assay of matrix metalloproteinase 9 (MMP9) has attracted considerable attentions for the clinical diagnosis of disease in early stage. In this report, we covalently engineer a fluorescein isothiocyanate-labeled peptide (Pep-FITC) linker containing the specific cleavage substrate of MMP9 onto the surface of hydrothermally reduced nano-graphene oxide (nrGO) to develop a FRETbased nrGO-Pep-FITC nanoprobe for ultrasensitive detection of MMP9. Upon cleavage of the Pep-FITC at the amide bond between Ser and Leu by MMP9, FITC was separated from nrGO, and fluorescence recovery of FITC was proportional to the MMP9 concentration within 0.01-0.06 nM and 0.06-0.15 nM ranges, respectively, in an aqueous solution, exhibiting 0.83 pM of detection limit. This nanoprobe is stable under borate buffer (pH = 7.4) with bovine serum albumin or 0.1% surfactant Triton 100 or Tween 20, and has good specificity for MMP9 detection, which is meaningful for MMP9-related clinical and bioanalytical applications.